The small nuclear RNAs (snRNAs) known as U1, U2, U3, U4, etc. comprise a highly abundant class of metabolically stable RNA molecules present in the nuclei of eukaryotic cells. Approximately 13 U-snRNAs have been identified in humans. In addition, at least one herpes virus (herpesvirus saimiri) has been shown to encode novel U-snRNAs expressed during viral infection of host cells. In vivo, the snRNAs exist as integral components of small nuclear ribonucleoprotein particles (snRNPs) which are recognized by antibodies from patients with certain autoimmune diseases. It is now well established that snRNPs play essential roles in messenger RNA (mRNA) and ribosomal RNA processing. Since the snRNAs have such fundamental roles in cellular metabolism, it is of interest to study the mechanisms involved in the expression of the genes that code for this particular class of small RNA molecules. The genes encoding the snRNAs are an unusual class of transcription units. With the exception of U6, they are synthesized by RNA polymerase II (Pol II), but they have promoter structures and cis-acting elements that functionally distinguish them from mRNA transcription units. For example, in the case of vertebrate snRNA genes transcribed by POl II, the transcription initiation site is specified by a conserved element lying about 50-60 base pairs upstream of the start site. Although much has been learned about the assembly of transcription pre-initiation complexes on mRNA promoters within the past few years, little is known about the protein factors involved in the assembly of Pol II transcription complexes on snRNA genes. We propose to identify, purify, and characterize these factors using biochemical and reverse genetic methods, placing particular emphasis upon factors unique to snRNA gene expression. Since vertebrate snRNA genes are transcribed poorly by Pol II in vitro, the accurate and highly efficient D. melanogaster in vitro system will be used to study the roles of individual factors and to investigate the molecular interactions that occur during transcription complex assembly on snRNA gene promoters. With the information obtained from Drosophila, vertebrate systems should become more amenable to study. Previous work from this lab has identified factors that bind to upstream regulatory (enhancer) elements that stimulate the basal level of chicken snRNA gene expression. Studies will be conducted to determine how these factors communicate with the basal machinery to enhance transcription complex assembly and/or initiation by Pol II. Finally, there are two U4 RNA genes in the chicken genome that code for different sequence variants of U4 RNA (U4B and U4X) that are differentially expressed as a function of development and tissue-type. Studies will continue into the molecular mechanisms that regulate the differential expression of these two U4 RNA genes, with particular emphasis places upon the role of a factor that binds specifically to the proximal regulatory region of the U4X gene.